Enzymatic and Chemical In Vitro Reconstitution of Iron-Sulfur Cluster Proteins

Iron-sulfur (Fe-S) clusters are key cofactors for proteins involved in essential cellular processes such as DNA replication and repair, ribosome biogenesis, tRNA thio-modification, and co-enzyme synthesis. Fe-S clusters can assemble spontaneously from inorganic compounds, but their biogenesis requires dedicated machineries to circumvent the toxic nature of iron and sulfur. To address how these machines work, different laboratories have applied various biochemical and biophysical approaches, both in vivo and in vitro. Fe-S cluster enzymatic and chemical formation in vitro is the most efficient way to follow Fe-S cluster biogenesis in a controlled environment and investigate each component of the machinery at the molecular level. In this review, we detail and discuss an efficient protocol for an in vitro Fe-S cluster enzymatic and chemical formation, which we successfully developed to study Fe-S cluster formation. We underline the applications of this approach to the study of an essential biological system.